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BIOC2004 CH > 1. Protein Structure and Folding > Flashcards

1. Protein Structure and Folding Flashcards

1
Q

What is the concentration of macromolecules in a typical cell?

A

400mg/ml

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2
Q

What is Lysozyme amyloidosis?

A

A form of systemic amyloidosis (where proteins adopt alternative conformations and form protein deposits) caused by misfolding of lysozyme

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3
Q

What are the three methods sued to determine 3D protein structure?

A

X-ray crystallography
Cryo-Electron microscopy
NMR spectroscopy

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4
Q

Describe Anfinsen’s experiment

A

An experiment used to determine protein folding. RNaseA was used (degrades RNA)

  1. Urea and mercaptoethanol were added to a native form of RNaseA which was catalytically active
  2. The reducing agents cause disulphide cross links to reduce and the protein to unfold. This caused catalytic inactivation of RNaseA
  3. Urea/Mercaptoethanol were then removed. RNaseA became catalytically again again as the disulphide cross links were reformed
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5
Q

What contains the information retried for polypeptide chain folding?

A

All information contained in the primary structure (AA sequence)

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6
Q

Why do proteins have a fine balance between folded and unfolded states? What does this enable to do (5)?

A

A fine balance is required as proteins are required for:

  • controlling their own availability and those of other proteins
  • precise timing of cellular events such as signalling and transport
  • must be able to degrade and create proteins easily
  • Fast turnover
  • Some enzymes need structural flexibility
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7
Q

How do proteins fold (roughly)?

A

Due to the hydrophobic effect. Non-polar residues (A, V, L, I, F, W, M) bury within the proteins structure, away from the watery cytosol etc. This forms a hydrophobic core within the protein. Polar/charged side chains remain on the surface and interact with water

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8
Q

How fast does protein folding occur?

A

Less than a second

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9
Q

What is the protein folding problem?

A

Protein can fold into many many structures, would take ages to find the right conformation

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10
Q

What are the three classical mechanisms for describing protein folding?

A
  1. Framework model - local elements of native secondary structure form, elements collide to form tertiary structure
  2. Hydrophobic collapse model - protein collapses around h’phobic side chains (molten globule) then side chains rearrange to final structure
  3. Nucleation-condensation model - well defined intermediate with flickering native structure h
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11
Q

What is a folding funnel?

A

A method of describing the folding of proteins, showing how the protein goes from many random conformations to the native conformation.

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12
Q

What is a protein’s ensemble?

A

The proteins state

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13
Q

If a protein does not fold spontaneously, how may it fold?

A

Using chaperones:

Class I: HSP70 type chaperones (HSP7-, HSP40, DnaK, DnaJ)

Class II: Chaperonins (GroEL, GroES)

These also help protein folding:

Protein disulphide isomerase

Peptide prolyl cis-trans isomerase

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14
Q

What disease may protein misfolding cause?

A

Parkinsons (alpha synuclein), Alzheimers (beta amyloid), ALS (SOD, TDP-43)

Cystic fibrosis - CFTR misfolding

Type II diabetes - amylin

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15
Q

Where will charged and polar amino acids be on a protein?

A

Map to protein surfaces, non-polar AA buried in cores of proteins

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16
Q

What is the name for a cluster of conserved residues?

A

Motifs

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17
Q

How would you find conserved residues?

A

AA Sequence Alignment

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18
Q

What 4 things make up an AA?

A

alpha amino group, R-group, alpha carbon (chiral centre) and alpha carboxyl group

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19
Q

What are the three classes of AA? Which AA are in each?

A

Non-polar - G, P, L, S, A, F, M, C, V, W, I

Polar non-charged - T, N, Y, Q

Charged - D, E (-ve), K, R, H (+ve)

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20
Q

How are polypeptide chains assembled?

A

Condensation reaction between OH of carboxyl and H of amino group. Iterative process to make polypeptide chain.

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21
Q

How do you determine whether an AA is in it’s D or L form?

A

CORN rule. If you look t the AA with R group pointing up then if it is L-form it will spell CO-R-N

L-CORN!!!

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22
Q

How do AA exist in 3D space?

A

Tetrahedral

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23
Q

What two types of bond orientation exist between peptides? Which is the most common?

A

Cis and trans. Trans is the most common due to the steric interactions between R-groups

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24
Q

Which AA does not favour the trans peptide bond as much? Why not?

A

Proline (20% cis) - due to the pyridine ring (?) - less steric hindrance is cis form from R groups

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25
Q

What is a dihedral/torsion angle?

A

The angle of twist between about the bonds B-C in an AA chain of A-B-C-D.

Angle of rotation needed to make the projection of the line B-A coincide with the projection of the line C-D.

Positive is clockwise

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26
Q

What are the angles phi and psi?

A

Phi = torsion angle between alpha carbon and nitrogen

Psi = torsion angle between alpha carbon and C

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27
Q

What are the rough phi/psi angles for beta sheets and alpha helices?

A

Beta sheets: -ve phi angle, +ve psi angle

alpha helices: -ve phi angle- -ve psi angle

28
Q

What rotamers do side chains favour?

A

Not staggered (eclipsed) or staggered

29
Q

Describe the structure of the alpha helix. What two forms of the alpha helix are there?

A

Carbonyl oxygen of residue i forms a H-bond with amide of residue i+4.

Side chains of AA project away from helix

3,10 helix - overwound
pi helix - underwound

30
Q

What is the helical wheel?

A

A method of describing helices, looks like a coil of rope kinda

Enables you to see whether AA are buried, partially exposed, or exposed

31
Q

How are helices stabilised?

A

(Large Macroscopic) Helix dipole: All peptide dipoles are orientated in the same direction giving the alpha helices a net dipole moment. Net bond polarisation stabilises coordination of negatively charged groups at N end

32
Q

Describe the structure of the beta sheet

A

Carbonyl oxygens and amides for, hydrogen bonds

May be antiparallel or parallel, planar or twisted

Alternate side chains along stand point to either side of the sheet, side chains on neighbouring strands point to the same side

33
Q

How are strands in anti-parallel sheets connected?

A

With short loops

34
Q

What is circular dichroism analysis?

A

A measure of the molar absorptivity differences of left and right circularly polarised light. Characteristic signals are produced with allow chain conformation of peptides to be determined

35
Q

Describe the beta turn

A
  • 180deg. turn
  • H-bond between i (carboxyl) and i+3 (amide)
  • P in 2 and G in 3 are common residues
  • used when beta sheets change direction
36
Q

What is a ramachandran plot?

A

A plot which compares the phi and psi angles of the polypeptide secondary structure.

It may be used to validate protein structures which have been determined by X-ray crystallography.

37
Q

What is the usual tertiary structure of a protein?

A

Globular

38
Q

What stabilises the tertiary structure of a protein?

A
  • Hydrogen bonds - weak
  • Ionic/electrostatic bonds - very weak
  • Disulphide bonds - very strong (covalent, reversible)
  • Hydrophobic force
39
Q

Give an example of 2 enzymes which require metal ions

A

Ribonucleotide reductase - Fe

Alcohol dehydrogenase - Zn

40
Q

How is Zn(II) bound in Carbonic anhydrase?

A

Zn(II) is coordinated by 3 His residues and 1 hydroxide residue

41
Q

What is the typical role of a Zinc Finger motif? What is the role of the Zinc?

A

Important for DNA binding in transcription factors.

The Zn(II) is essential for the ability of the tightly folded protein chain

42
Q

What are the three main categories of tertiary structure motifs? Give examples of each

A

All alpha - EF hand, 4 helix bundle

All beta - beta barrel, beta hairpin, greek key

Alpha/Beta - TIM barrel, Rossman fold, beta-alpha-beta

43
Q

Describer the structure of the EF hand? Where is it used?

A

All alpha motif consisting of 2 helices joined together in fixed conformation. Binds calcium.

Used in Signalling proteins such as calmodulin as capable of large conformational change

44
Q

Describe the beta sandwich structure. Where may it be used in?

A

Two beta sheets held together.

Found in immunoglobulin like proteins (antibodies) and Titin (muscle ennuit)

45
Q

How are the beta strands arranged in beta barrels?

A

Antiparallel

46
Q

What motif is present in bovine trypsin inhibitor and snake venom erabutoxin?

A

Beta-hairpin

47
Q

Describe the greek key

A

Extension of a beta hairpin, consists of 4 adjacent antiparallel beta strands linked together by short loops

No particular function but exists nonetheless (like wasps)

48
Q

What are the two different forms of the beta-alpha-beta motif?

A

Right handed (helix above), left-handed (helix below)

49
Q

How are the TIM barrel and Rossman fold similar? How do they differ? Give examples of proteins which contain these motifs

A

Both made up from beta-alpha-beta motifs

TIM barrel all the b-a-b motifs are connected in sequence and all the helices are above the sheet, whereas in the Rossman fold there are loops at COOH terminal diverge, the helices are present on both sides of the sheet (enables binding)

TIM: Triose phosphate isomerase
Rossman: Alcohol dehydrogenase

50
Q

Describe the active site of the Rossman fold

A

Alpha helices occur either side of a parallel/mixed beta sheet. This forms a crevice where the loops at the carboxy terminal end of the diverge.

51
Q

What is a mosaic structure?

A

A large protein which is made up multiple domains (multiple motifs make up domains)

52
Q

What is a domain?

A

The packing together of structural motifs to form stable, independent units of tertiary structure - conserved within related proteins

53
Q

What is the size range of a domain?

A

90% are no more than 200AA, avg. ~100AA

54
Q

Give an example of a single domain and multodmain protein

A

Single domain: GFP

Multi domain: Firefly luciferase

55
Q

What is a knot?

A

A polypeptide chain which is you pulled the two ends apart it would form a knot (rather than completely unravel)

56
Q

What are IDPs? What do they tend to have?

A

Intrinsically Disordered Proteins - no defined 2D or 3D structure

Have fewer hydrophobic residues (smaller hydrophobic effect) and a higher amount of polar and charged residues

57
Q

How does the folding funnel of an IDP vary from an ordered protein?

A

The IDP folding funnel will be flat and wiggly, more like ECG output than a an upside down mountain

58
Q

Do IDPs have any structure?

A

Yes, they often contain helices

59
Q

What is the role of IDPs?

A

Mediation of interactions (gain structure when bound to target)

60
Q

Give 4 examples of IDP roles in the body

A

Alpha synuclein- form structure upon interaction with calmodulin/lipid membranes

p53 - IDPs mediate p53 interactions

BRCA1 - involved in biological signal processes (DNA damage, apoptosis…)

prion proteins - protein infections - fuck shit up

61
Q

What is the benefit of the IDPs?

A

The amount of disorder increases the function space and means that the IDPs can interact with a wider variety of different partners

62
Q

How does alpha-synuclein change when it binds calmodulin?

A

The alpha synuclein binds to calmodulin, causing a helical structure to form at the N-terminus. Negatively regulates calmodulin

63
Q

How does alpha-synuclein lead to parkinson’s?

A
  • mutation in A30P, A53T, E46Q and gene triplication
  • cause A-syn (usually bound to membrane/proteins) to detach
  • forms a pre fibrillar species in the cytoplasm
  • amyloid fibrils are then formed
  • these then form lewy bodies which cause parkinson’s
64
Q

Describe the structure of GroEL/ES

A

GroEL: 2 seven-membered rings stacked on top of each other, each component subunit consists of an apical, intermediate and equatorial domain (x7 = ring)

GroES: little hat to go on top

65
Q

Describe the function of GroEL/ES

A

Chaperonin: Assists in protein folding & assembly

  1. Partially folded protein enters GroEL cage, binds to h’phobic surface near rim
  2. ATP is used to bind GroES to capture protein
  3. GroEL double in volume and becomes hydrophilic - favouring protein folding
  4. ATP hydrolysed, GroES dissociates causing protein release
  5. If protein is not completely folded then may reenter GroEL for another round ;)
66
Q

What is the function of the apical, intermediate and equatorial domains of the component subunit of GroEL/ES?

A

Apical - protein and GroES binding

Intermediate - hinge connects apical and equatorial covalently

Equatorial - mediates ATP binding

67
Q

How does GroEL/ES fold a large protein?

A

trans folding:

  1. protein binds to open GroEL cavity, GroES released from opposite end
  2. GroES and ATP re-bind to GroEL and protein is released

BIOC2004 CH (11 decks)

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